Oligonucleotide probes, kit containing the same and method for pathotyping of h5 avian influenza viruses

ABSTRACT

The present invention provides a group of specific probes directed to cleavage site of hemagglutinin precursor protein of avian influenza virus subtypes H5, and provides a method for rapid pathotyping of H5 avian influenza virus. The present invention further provides a kit containing the probes and the kit is easy-to-use, low-cost, high sensitivity, enabled the molecular pathotyping of H5 viruses by a simpler and faster means that conventional methods.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to detection of infectious agents, morespecifically, by using oligonucleotide probes, kit containing the samefor rapid pathotyping of H5 avian influenza viruses.

2. Description of the Related Art

Avian Influenza virus (AIV) infects many animals such as humans, pigs,horses, marine mammals, and birds. Its natural reservoir is in aquaticbirds, and in avian species most influenza virus infections cause mildlocalized infections of the respiratory and intestinal tract. While themajority of AIV subtypes are classified as low-pathogenicity avianinfluenza viruses (LPAIV), the H5 and H7 subtypes have the ability tomutate to high pathogenic avian influenza viruses (HPAIV). The HPAIV canhave high pathogenic effect in poultry, with sudden outbreaks causinghigh mortality rates in affected poultry populations. In humans, theHPAIV cause a highly contagious acute respiratory disease that haveresulted in epidemic and pandemic disease. Thus, it is of greatimportance to rapidly detect influenza viruses and promptly discriminatebetween LPAIV and HPAIV.

Reliable methods for pathotyping of AIV H5 viruses are based ondetermination of the intravenous pathogenicity index (IVPI) in specificpathogen free (SPF) chickens and on characterization of thehemagglutinin (HA) gene cleavage site (SC) by sequencing. The amino acidmotif at the CS of the HA precursor protein of H5 viruses has been foundto have a consistent format, PQ^(m)-X^(n)-*GLF (SEQ ID NO: 51) (Leijonet al., Journal of Clinical Microbiology, November 2011, p. 3860-3873)and a large number of Genbank nucleotide analysis (^(m) Glutaminecomposes the vast majority, although other residues may be seen but few.X: all kinds of residues, in the present invention, it specificallyrepresents arginine and lysine; n: the number of X. *Actual cleavagesite at HA₀ by host proteases.) The number of basic amino acids(arginine and lysine) at X is an indicator of pathogenicity; in general,LPAIVs have one to three basic amino acids (n=1˜3), and HPAIVs have fouror more basic amino acids (n=4 or more).

Currently, there are a variety of techniques that can be used as apathogenicity test for H5 avian influenza virus in biological samples,including PCR followed by nucleotides sequencing across the CS,real-time PCR, restriction enzyme cleavage pattern of reversetranscription PCR product and the like. However, those methods aretime-consuming, labor-intensive, costly and highly complicated toperform. A microarray system, ArayTube, has been reported to pathotypeAIV, however, the result interpretation needed the reader and was onlylimited to parts of H5 viruses, e.g. highly pathogenic H5/N1/Aisa clade2.2.

Therefore, there remains a need for a lower cost, easier implement, morecomprehensive and rapid approach with high sensitivity and specificity.

SUMMARY OF THE INVENTION

One aspect of the invention is to provide a probe selected from thegroup consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.

Another aspect of the invention is to provide a kit for pathotyping ofH5 avian influenza virus, comprising the probe selected from the groupconsisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.

Preferably, the probe is further added a tail composed of 19 T bases andis spotted to a microarray substrate.

Preferably, the kit further comprising an oligonucleotide of SEQ ID NO:14 spotted on the microarray substrate as a positive control probe.

Still another aspect of the invention is to provide a method forpathotyping of H5 avian influenza virus in a sample, comprising: (a)obtaining a nucleic acid from the sample; (b) hybridizing the nucleicacid with a probe selected form the group consisting of: SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12 and SEQ ID NO: 13; and (c) pathotyping the sample as alow-pathogenic avian influenza virus (LPAIV) or highly pathogenic avianinfluenza virus (HPAIV) based on the hybridization result.

Preferably, the nucleic acid is obtained by extracting RNA from thesample, reverse transcribing the RNA into a DNA, and amplifying the DNAby a PCR reaction.

Preferably, a set of primers of SEQ ID NO: 15 and SEQ ID NO: 16 are usedin the PCR reaction.

Preferably, the set of primers is 5′ end-biotinylated.

Preferably, the nucleic acid further comprises a label. Preferably, thelabel is a fluorescent label, a chemiluminescent label, a colored dyelabel, a radioactive label, a radiopaque label, a protein including anenzyme, a peptide or a ligand.

Preferably, the H5 avian influenza virus is a highly pathogenic avianinfluenza viruses (HPAIV) when the nucleic acid is hybridized to theprobe selected from SEQ ID NO: 12 and SEQ ID NO: 13 or the nucleic acidis hybridized only to the probe of SEQ ID NO: 1 or SEQ ID NO: 2, or bothwhen all the probes are used.

Preferably, the H5 avian influenza virus is a low pathogenic avianinfluenza viruses (LPAIV) when the nucleic acid is hybridized to theprobe selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, SEQ ID NO: 10 and SEQ IDNO: 11.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting and non-exhaustive embodiments will be described inconjunction with the accompanying drawings. Understanding that thesedrawings depict only several embodiments in accordance with thedisclosure and are, therefore, not to be intended to limit its scope,the disclosure will be described with specificity and detail through useof the accompanying drawings, in which:

FIG. 1 shows detection and differentiation of AIVs using oligonucleotidemicroarrays. (A) Microarray map. The meaning of each probe and itsdetecting strains are shown in Table 3. P: positive control. Themicroarray detection results of LPAIVs, HPAIVs and the artificialoligonucleotides with five or more basic amino acids at the CS are shownon (B), (C), and (D), respectively. The denotation of each artificialoligonucleotide is shown in Table 2.

FIG. 2 shows detection limit test using A/CK/Hsinchu/A1939/11 (LV7) asan example. (A) Cloned plasmid was serial diluted (indicated as copynumbers) and PCR-amplified using primer pair H5f/H5r. M: 100 bp laddermarker; 1: 4.6×10⁷ copies/μL; 2: 4.6×10⁶ copies/μL; 3: 4.6×10⁵copies/μL; 4: 4.6×10⁴ copies/μL; 5: 4.6×10³ copies/μL; 6: 4.6×10²copies/μL; 7: 4.6×10¹ copies/μL; 8: 4.6×10° copies/μL; 9: negativecontrol. (B) The corresponding results on the oligonucleotidemicroarrays.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, illustrative embodiments and examples of the presentdisclosure will be described in detail with reference to theaccompanying drawings so that inventive concept may be readilyimplemented by those skilled in the art.

However, it is to be noted that the present disclosure is not limited tothe illustrative embodiments but can be realized in various other ways.In the drawings, certain parts not directly relevant to the descriptionare omitted to enhance the clarity of the drawings, and like referencenumerals denote like parts throughout the whole document.

Throughout the whole document, the term “comprises or includes” and/or“comprising or including” used in the document means that one or moreother components, steps, operations, and/or the existence or addition ofelements are not excluded in addition to the described components,steps, operations and/or elements. The terms “about or approximately” or“substantially” are intended to have meanings close to numerical valuesor ranges specified with an allowable error and intended to preventaccurate or absolute numerical values disclosed for understanding of thepresent invention from being illegally or unfairly used by anyunconscionable third party. The terms “a” and “an” refer to one or tomore than one (i.e., to at least one) of the grammatical object of thearticle.

The present invention provides oligonucleotide probes, kit containingthe oligonucleotide probes and method for rapid pathotyping of H5 avianinfluenza viruses.

[Probes for Pathotyping of H5 Avian Influenza Viruses]

The present invention provides a probe selected from the groupconsisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 forpathotyping of H5 avian influenza viruses as a low-pathogenic avianinfluenza virus (LPAIV) or highly pathogenic avian influenza virus(HPAIV).

The term “pathotyping of H5 avian influenza viruses” used herein refersto detection of the number of X in the consistent format,PQ^(m)-X^(n)-*GLF (SEQ ID NO: 51), in H5 AIV amino acid motif at the CSof the HA precursor protein, wherein X represents basic amino acids(arginine (R) and lysine (K)). The term “low-pathogenic avian influenzavirus (LPAIV)” used herein refers to H5 AIV has one to three basic aminoacids at the above motioned amino acid motif, and the term “highlypathogenic avian influenza virus (HPAIV)” refers to H5 AIV has four ormore basic amino acids at the above motioned amino acid motif.

In the present invention, the term “probe” includes at least 10nucleotides of the nucleic acid sequences that are shown to encodespecific amino acids or proteins. When referring to a probe, the term“specific for (a target sequence)” indicates that the probe hybridizesunder stringent conditions substantially only to the target sequence ina given sample comprising the target sequence. The term “hybridization”used herein refers to the process wherein oligonucleotides and/or theiranalogs bind by hydrogen bonding, which includes Watson-Crick, Hoogsteenor reversed Hoogsteen hydrogen bonding, between complementary bases.Generally, nucleic acid consists of nitrogenous bases that are eitherpyrimidines (Cytosine (C), uracil (U), and thymine (T) or purines(adenine (A) and guanine (G)). These nitrogenous bases form hydrogenbonds consisting of a pyrimidine bonded to a purine, and the bonding ofthe pyrimidine to the purine is referred to as “base pairing.” Morespecifically, A will bond to T or U, and G will bond to C.“Complementary” refers to the base pairing that occurs between twodistinct nucleic acid sequences or two distinct regions of the samenucleic acid sequence. A person skilled in the art will appreciate that,depending on the context, the terms “binding”, “hybridizing” or“hybridization” may be used interchangeably without giving rise toambiguity. In the present invention, the probe may have the nucleotidesequence ambiguity. Where a nucleotide position is ambiguous and may berepresented by one or more nucleotides, standardized symbols or letters,well known to a person skilled in the art, as given in the sequencelisting of this application are used. Such symbols or letters, proposedby the International Union of Pure and Applied Chemistry which alsocorresponds to WIPO Standard ST.25 Appendix 2 Table 1, are as follows: Mis A or C; R is A or G; W is A or T; S is C or G; Y is C or T; K is G orT; V is A or C or G; H is A or C or T; D is A or G or T; B is C or G orT; N is G or A or T or C.

In the present invention, the probes are used to detect and discriminatethe basic amino acid number at the X of the PQ^(m)-X^(n)-*GLF (SEQ IDNO: 51) motif at the CS of the HA precursor protein of the H5 viruses.In the present invention, the probe selected from SEQ ID NO: 3 and SEQID NO: 4 detects LPAIV having 1 basic amino acid at X site (n=1); theprobe selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8 and SEQ ID NO: 9 detects LPAIV having 2 basic amino acids at X site(n=2); the probe selected from SEQ ID NO: 10 and SEQ ID NO: 11 detectsLPAIV having 3 basic amino acids at X site (n=3); the probe selectedfrom SEQ ID NO: 12 and SEQ ID NO: 13 detects HPAIV having 4 basic aminoacids at X site (n=4). The probe, SEQ ID NO: 1 or SEQ ID NO: 2, is auniversal probes, and all AIV H5 viruses can hybridize with either SEQID NO: 1 or SEQ ID NO: 2, or both. Thus, when all of the 13 probes (SEQID NO: 1 to SEQ ID NO: 13) are used, the sample of AIV H5 virus possessfive or more basic amino acid (categorized as HPAIV) at the X (n=5) ishybridized either SEQ ID NO: 1 or SEQ ID NO: 2, or both, but is nothybridized with the probes of SEQ ID NO: 3 to SEQ ID NO: 13 whichdetects 1 to 4 basic amino acid(s) at X site (=1 to 4).

[Method for Rapid Pathotyping of H5 Avian Influenza Viruses]

According to the present invention, a method for pathotyping of H5 avianinfluenza virus in a sample is provided. The method comprises: (a)obtaining a nucleic acid from the sample; (b) hybridizing the nucleicacid with a probe selected form the group consisting of: SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12 and SEQ ID NO: 13; and (c) pathotyping the sample as aLPAIV or HPAIV based on the hybridization result.

According to the present invention, the term “sample” may be abiological sample, for example any sample collected from an individualsuspected of carrying avian influenza virus subtype H5 or H5N1. Thesample may be any sample that contains the virus from an infectedindividual, and includes tissue and fluid samples, for example, blood,serum, plasma, peripheral blood cells including lymphocytes andmononuclear cells, sputum, mucous, urine, feces, throat swab samples,dermal lesion swab samples, cerebrospinal fluids, pus, and tissueincluding spleen, kidney and liver.

According to the present invention, a nucleic acid from the sample isextracted from the sample for further evaluation. Preferably, a RNA fromthe sample is first extracted, and then reverse transcribed into a DNA(namely, cDNA), and the DNA is further amplified by a PCR reaction.

The term “RNA” refers to a sequence of two or more covalently bonded,naturally occurring or modified ribonucleotides. The RNA may be singlestranded or double stranded. The term “DNA” refers to a sequence of twoor more covalently bonded, naturally occurring or modifieddeoxyribonucleotides, including cDNA and synthetic (e. g., chemicallysynthesized) DNA, and may be double stranded or single stranded. By“reverse transcribed DNA” or “DNA reverse transcribed from” is meantcomplementary or copy DNA (cDNA) produced from an RNA template by theaction of RNA-dependent DNA polymerase (reverse transcriptase). Thetemplate RNA for the reverse transcription reaction may be obtained froma sample using RNA extraction methods known in the art. RNA extractionkits are also commercially available, for example, RNeasy kits (Qiagen),and the availability and use of such kits will be known and understoodby a skilled person.

Once the reverse transcription reaction is completed, thesingle-stranded DNA molecule can be used in the amplification reaction.The term “amplifying” or “amplification” refers to a reaction in which anucleic acid molecule that is to be detected so as to pathotyping of H5avian influenza virus as a HPAIV or a LPAIV. A suitable polymeraseenzyme will be used to synthesize a new strand of a template nucleicacid to generate multiple copies.

The amplification step may be performed in the same reaction as thereverse transcription reaction, provided the conditions and reagentsfrom the reverse transcription do not interfere with the amplificationreaction. Alternatively, the reverse transcription product may bepurified prior to being used as template in the amplification reaction.

According to the present invention, amplification is performed by a PCRamplification reaction. Thus, the amplification step may be performedwith a DNA polymerase, for example, Taq polymerase, using standardmethods and techniques that are known to a person skilled in the art.DNA polymerases for use in amplification of DNA molecules arecommercially available. The amplification reaction is performed underconditions and with the necessary reagents, such as deoxynucleotides,buffer and relevant forward and reverse primers, so as to optimize thepolymerization activity of the DNA polymerase enzyme.

The PCR amplification reaction involves a denaturation step, in whichthe reaction is heated to a temperature sufficient to denature thetranscribed DNA strand and to prevent binding of the primers to eitherstrand. The denaturation step is followed by an annealing step, in whichthe reaction temperature is ramped down to a temperature at which theprimers can bind to the DNA strand. The final step is an extension step,in which the reaction is heated to a temperature that is optimal forextension of the primer by the DNA polymerase. These three steps arecycled through multiple times allowing for the production of thecomplementary strand of DNA that pairs with the reverse transcribed DNAand of the reverse transcribed strand by extension from the forward andreverse primer or primers respectively. In each successive round of theamplification reaction, more of each DNA strand is produced, which thenmay be used as template for the next cycle, resulting in amplificationof the DNA product. A skilled person can readily determine theappropriate temperature for each segment of the amplification step andthe desired number of cycles to be performed.

According to the present invention, the PCR procedure amplifies aconserved region of the CS of the HA precursor protein among HPAI andLPAI strains using specific primers.

As will be understood by a skilled person, a “primer” is asingle-stranded DNA or RNA molecule of defined sequence that can basepair to a second DNA or RNA molecule that contains a complementarysequence (the target). The stability of the resulting hybrid moleculedepends upon the extent of the base pairing that occurs, and is affectedby parameters such as the degree of complementarity between the primerand target molecule and the degree of stringency of the hybridizationconditions. The degree of hybridization stringency is affected byparameters such as the temperature, salt concentration, andconcentration of organic molecules, such as formamide, and may bedetermined using methods that are known to those skilled in the art.Preferably, the primer may be modified with a label to allow fordetection of the primer or a DNA product synthesized or extended fromthe primer. For example, the label may be a fluorescent label, achemiluminescent label, a coloured dye label, a radioactive label, aradiopaque label, a protein including an enzyme, a peptide or a ligandfor example biotin. Preferably, the primer is 5′ end-biotinylated sothat the yielded PCR products are biotinylated and are around 440 bp.

According to the present invention, since the PCR product (which is alsoknown as “nucleic acid” as defined herein) is labeled when synthesizedin the PCR procedure using labeled primers, the PCR product may befurther detected by nucleic acid hybridization methods, single strandedconformational polymorphism (SSCP) analysis, restriction fragmentpolymorphism (RFLP) analysis, southern hybridization, northernhybridization, in situ hybridization, electrophoretic mobility shiftassay (EMSA), nucleic acid microarrays, and other methods that are knownto those skilled in the art.

According to the present invention, the labeled PCR product (nucleicacid) is hybridizing with a probe selected form the group consisting of:SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 for differentiating as aLPAIV or HPAIV based on the hybridization result. As described above,the sample contains HPAIV when the nucleic acid is hybridized to theprobe selected from SEQ ID NO: 12 and SEQ ID NO: 13 or the nucleic acidis hybridized only to the probe of SEQ ID NO: 1 or SEQ ID NO: 2, or bothwhen all the probes are used; the sample contains LPAIV when the nucleicacid is hybridized to the probe selected from SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10 and SEQ ID NO: 11.

The method for detection with the hybridization result based ondifferent labeling of the PCR product is well-known in the art.Preferably, the PCR products are biotinylated so that the hybridizationresult is detected by the Biotin-Streptavidin System.

[Kit Containing the Oligonucleotide Probes]

The present invention also generally relates to a kit for pathotyping ofH5 avian influenza virus in a biological sample or from biologicalmaterial isolated and/or purified from a biological sample/

According to the present invention, a kit for pathotyping of H5 avianinfluenza virus, comprising the probe selected from the group consistingof: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, is provided.Preferably, the probe may be immobilized on a solid support usingstandard methods, including chemical cross-linking, photocross-inking,or specific immobilization via a functional group on the probe.According to the present invention, a 19 T bases are added on each 5′end of probes and each probe is then spotted on the microarray polymersubstrate using automatic spotting machine and immobilized by a UVcrosslinker. The kit according to the present invention may optionallyinclude a positive control probe, for example, a nuclei acid or at leastportion thereof. Preferably, an oligonucleotide of SEQ ID NO: 14 isspotted on the microarray substrate as a positive control probe. The kitmay further comprise information for use of the kit. It may be forexample illustrative information provided by the manufacturer.

EXAMPLE

Hereinafter, the present disclosure will be specifically described withreference to examples and drawings. However, the present disclosure isnot limited to the examples and the drawings.

[Origin of Influenza Virus Reference Strains and Field Samples]

The AIV reference strains used in the present invention are listed inTable 1. LV1-LV3 were field isolates originated from the GraduateInstitute of Veterinary Medicine, National Taiwan University. LV4-LV10and HV1-HV5 were obtained from the Epidemiology Division of the AnimalHealth Research Institute, Council of Agriculture, Tamsui, Taiwan.

TABLE 1 AIV strains used in the present invention Serial Genbank no.Strain designation Subtype pathogenicity* acc. no. LV1 A/duck/Yunlin/04H5N2 LP LV2 A/duck/Hong Kong/820/80 H5N3 LP LV3 A/chicken/Taiwan/1209/03H5N2 LP AY573917 LV4 A/CK/Taiwan/1103/12 H5N2 LP LV5A/CK/Changhua/0102/10 H5N2 LP LV6 A/CK/Chiayi/A1625/11 H5N2 LP LV7A/CK/Hsinchu/A1939/11 H5N2 LP LV8 A/CK/Nantou/A2085/12 H5N2 LP LV9A/CK/Penghu/A2721/12 H5N2 LP LV10 A/CK/Miaoli/1203/12 H5N2 HP HV1A/CK/Changhua/A1029/10 H5N2 HP HV2 A/CK/Changhua/A1321/10 H5N2 HP HV3A/CK/Changhua/0101/12 H5N2 HP KF193386 HV4 A/CK/Changhua/0302/12 H5N2 HPHV5 A/CK/Yunlin/0502/12 H5N2 HP *LP: low-pathogenicity; HP:high-pathogenicity

[RNA Extraction, Reverse Transcription and PCR]

Viruses were grown in the allantoic cavities of 9- to 10-day-oldembryonated fowl eggs originating from a commercial SPF flock. Viral RNAwas extracted from allantoic fluid using QIAamp Viral RBA kit (Qiagen,Valencia, Calif.). Field tissue samples were ground in liquid nitrogenand RNA was extracted us QIAamp RNeasy Mini Kit (Qiagen, Hilden,Germany). The reverse transcription was then performed with uni12 primer(Hoffmann B, Harder T, Starick E, et al.: 2007, J Clin Microbiol 45:600-603) using Transcriptor High Fidelity cDNA Synthesis Kit (Roche,Mannheim, Germany) according to the manufacture's instructions. SpecificH5 primer pair (H5f/H5r) flanking the CS was designed from conservedregions among HPAI and LPAI strains obtained from the GenBank data. Theprimer H5f (5′-ATWGCTCCNGAATATGCATWWAARA-3′ (SEQ ID NO: 15)) and H5r(5′-TCRAAYTGARTGTTCATTTTRTCAATG-3′ (SEQ ID NO: 16)) were 5′end-biotinylated and yielded products were around 440 bp. The PCR wascarried out in a reaction volume of 50 μl containing 5 μl of template, 5μl of each primer (10 μM), 5 μl of 10×PCR buffer, 4 μl of each dNTP (2.5mM), 0.3 μl (5 U/μl) of GenTaq Plus DNA polymerase (GeneMark, Taichung,Taiwan). The thermal profile for amplification was 94° C. for 3 min,40×(94° C. for 30 s, 50° C. for 30 s, and 72° C. for 40 s), 72° C. for 7min. Ten μl PCR products were separated in 1.5% agarose gels (Gibco,Grand Island, N.Y.), run in 0.5×TAE buffer with 0.5 μg/ml ethidiumbromide (Gibco, Grand Island, N.Y.) at 100 V for 40 min, and visualizedunder UV light.

[Cloning and Sequencing]

The PCR products were purified by PCR clean-up kit (GeneMark, Taichung,Taiwan) and cloned into plasmid vectors using pGEM-T EASY Vector System(Promega, Madison, Wis.) following the manufacturer's instructions. Theplasmids were then transformed into competent cells (ARROWTEC, Taipei,Taiwan) which were further cultured on the LB agar (BD Difco, Sparks,Md.). The colonies with successful inserts were confirmed by PCR usingplasmid T7 and SP6 primers and then amplified in the LB broth (BD Difco,Sparks, Md.). Plasmid DNA was extracted using Miniprep Purification Kit(Protech Co., Taipei, Taiwan) and the inserts were further sequenced bymeans of commercial service (Protech Co., Taipei, Taiwan). The HA₀nucleotide sequence spanning the cleavage site of each reference virusand the test sample was then attained.

[Artificial Oligonucleotides]

Because of the extensive nucleotide variety at the CS and the viralterritorial characteristic of the H5 AIVs, no virus isolates with fiveor more basic amino acids at the X of the PQ^(m)-X^(n)-*GLF (SEQ ID NO:51) motif were available in Taiwan. A comprehensive alignment andanalysis of all AIV H5 sequences from Genbank were made. Ninerepresentative sequences which possess five or more basic amino acids atthe X and encompass the CS diversity were selected. Oligonucleotidesspanning the CS region with 114-122 bases long of the ninerepresentative sequences were synthesized artificially (Table 2). Theseartificial oligonucleotides were amplified using their own primers inaccordance with the sequences located at the two ends. The ampliconswere verified on agarose gel and were further tested on the microarraysto simulate those genuine viruses.

TABLE 2Artificial oligonucleotides which possess five or more corresponding basic amino acids at theX of the PQ-X^(n)-GLF (SEQ ID NO: 51) motif. Artificial CorrespondingBasic amino oligo- amino acids acid number Genbank nucleotide^(a)Sequence (5′-3′)^(b) around the CS^(c) at X^(c) acc.no. 5b/5CCCCTTACCATTGGAGAGTGTCCCAAATATGTCAAATCAAATAAACTGGTCC PQ-RRKKR-GLF 5AB241613 TTGCAACAGGACCGAGGAACGTGCCTCAAAGAAGAAAAAAAAGAGGTCTGTT(SEQ ID NO: 26) TGGAGCAAAAGC (SEQ ID NO: 17) 5b/6TCCTTTTACTATTGGGGAGTGCCCCAAGTATGTCAAATCGAAAAAACTAGTT PQ-RKRKTR-GLF 5AB558474 CTTGCAACAGGGCTAAGAAACGTACCCCAAAGAAAAAGAAAAACAAGAGGCC(SEQ ID NO: 27) TATTTGGAGCAATAGC (SEQ ID NO: 18) 5b/8CTCTCACCATCGGGGAATGCCCCAAATATGTGAAATCAAACAGATTAGTCCT PQ-IEGRRRKR-GLF 5HM583608 CGCGACTGGACTCAGAAATGCCCCCCAAATAGAGGGAAGAAGAAGAAAAAGA(SEQ ID NO: 28) GGACTATTTGGAGCCATA (SEQ ID NO: 19) 6b/6CTCTCACCATTGGGGAATGCCCCAGATACGTGAAATCAGATAGATTAGTCCT PQ-RRRKKR-GLF 6AJ305306 TGCGACTGGACTCAGGAATGTCCCTCAAAGAAGAAGAAAAAAAAGAGGACTA(SEQ ID NO: 29) TTTGGGGCTATAGC (SEQ ID NO: 20) 6b/7-1CTCTCACCATCGGGGAATGCCCCAAATATGTGAAATCAAACAGATTAGTCCT PQ-RERRKKR-GLF 6AM183676 TGCGACTGGGCTCAGAAATAGCCCTCAAAGAGAGAGAAGAAAAAAGAGAGGA(SEQ ID NO: 30) TTATTTGGAGCTATAGC (SEQ ID NO: 21) 6b/7-2CTCTCACCATCGGGGAATGCCCCAAATATGTGAAATCAAACAAATTGGTCCT PL-RERRRKR-GLF 6EF419243 TGCGACTGGGCTCAGAAATAGTCCTCTAAGAGAAAGAAGAAGAAAAAGAGGA(SEQ ID NO: 31) CTATTTGGAGCTATAGC (SEQ ID NO: 22) 6b/8CTCTCACCATCGGGGAATGCCCCAAATATGTGAAATCAAACAGATTAGTCCT PQ-RESRRKKR-GLF 6CY091963 TGCAACAGGACTCAGAAACAGCCCTCAAAGAGAGAGCAGAAGAAAAAAGAGA(SEQ ID NO: 32) GGACTATTTGGAGCTATAGC (SEQ ID NO: 23) 7b/8-1CTCTCACCATCGGAGAATGTCCCAAATATGTGAAATCAAACAAATTAGTCCT PQ-RERRRRKR-GLF 7KF169907 TGCGACTGGGCTCAGAAATAGTCCTCAAAGAGAGAGAAGAAGAAGAAAAAGA(SEQ ID NO: 33) GGACTGTTTGGAGCTATAGC (SEQ ID NO: 24) 7b/8-2CTCTCACCATCGGGGAATGCCCCAAATATGTGAAATCAAACAGATTAGTCCT  PQ-RERRRKKR-GLF 7CY091949 TGCGACTGGGCTCAGAAATAGCCCTCAAAGAGAGAGAAGAAGAAAAAAGAGA(SEQ ID NO: 34) GGACTATTTGGAGCTATAGC (SEQ ID NO: 25) ^(a)The symboln′b/n means there are n′ basic amino acids among the total n amino acidsat X. ^(b)The nucleotide bases corresponding to PQ-X^(n)-GLF (SEQ ID NO:51) amino acid motif at CS are underlined. ^(c)Basic amino acid at the Xis marked with a bottom line and the number is counted.

[Probe Design]

The universal probes targeting all AIV H5 subtypes were designed fromthe conserved sequences of the HA gene located within the H5f/H5ramplicons. All AIV H5 viruses can hybridize with either universal probeU1 or U2, or both. Probes for differentiating LPAIVs and HPAIVs weredesigned based on the basic amino acid number at the X of thePQ^(m)-X^(n)-*GLF (SEQ ID NO: 51) motif of the CS. Elevendifferentiating probes comprehensively encompassing the highly variableCS region of the H5 viruses were designed. The designs of all probes aswell as the primer pair H5f/H5r were derived from the alignment andanalyses of the nucleotide sequences retrieved from the enormous GenBankdata, and conducted by the MegAlign program (DNASTAR, Madison, Wis.,US). The probe sequences are listed in Table 3.

TABLE 3Probes used to detect and discriminate the basic amino acid number atthe X of the PQ-X^(n)-GLF (SEQ ID NO: 51) motif at CS of H5 viruses.Basic amino acid AIV strains Corresponding number at used in this ProbeSequence (5′-3′) amino acids† X^(b) study U1^(a) GAGTGYCCMAARTAYGTSAAAT(SEQ ID NO: 1) U2^(a) GAATGYCCCARATAYGTGAAAT (SEQ ID NO: 2) 1ACCY CAR ATA GAR ACA AGR PQ-IETR 1 LV1 (SEQ ID NO: 3) (SEQ ID NO: 35) 1BCCY CAR GGA GAR ACA AGR PQ-GETR 1 (SEQ ID NO: 4) (SEQ ID NO: 36) 2ACCY CAR AGA GAR ACR AGA PQ-RETR 2 LV2 (SEQ ID NO: 5) (SEQ ID NO: 37) 2BCCY CAA AAR GAA ACA ARA PQ-KETR 2 (SEQ ID NO: 6) (SEQ ID NO: 38),PQ-KETK (SEQ ID NO: 39) 2C CCY MAA ARA GAA RCA AGA PK-RETR 2(SEQ ID NO: 7) (SEQ ID NO: 40), PQ-KEAR (SEQ ID NO: 41) 2DCCY CAA AGA RCC ACA ARA PQ-RATR 2 (SEQ ID NO: 8) (SEQ ID NO: 42),PQ-RATK (SEQ ID NO: 43), PQ-RTTR (SEQ ID NO: 44) 2ECCA GAG AAT CCA AAG CCC PE-NPKPR 2 (SEQ ID NO: 9) (SEQ ID NO: 45) 3AMGA GAA AAA AGA GGM CTA REKR-GL 3 LV3-LV10 (SEQ ID NO: 10)(SEQ ID NO: 46) 3B CCY CAA AGA AAA ACA AGA PQ-RKTR 3 (SEQ ID NO: 11)(SEQ ID NO: 47) 4A CCY CAA AGR ARR AAA AGA PQ-RKKR 4 HV1-HV3(SEQ ID NO: 12) (SEQ ID NO: 48) HV4-HV5 PQ-RRKR (SEQ ID NO: 49) 4BCCY CAG AAG AAR AAG AGA PQ-KKKR 4 (SEQ ID NO: 13) (SEQ ID NO: 50)^(a)Universal probes. All AIV H5 viruses can hybridize with either probeU1 or U2, or both. ^(b)Basic amino acid at the X is marked with a bottomline and the number is counted.

[Oligonucleotide Microarray Preparation and Hybridization Reaction]

A tail composed of 19 T bases was added on each 5′ end ofoligonucleotide probe, including the positive control probe (anoligonucleotide from capsid protein VP 1 of human enterovirus 71 gene,5′-ATGAAGCATGTCAGGGCTTGGATACCTCG-3′ (SEQ ID NO: 14)). Fifteen mM of eachprobe was then spotted to each specific position on the microarraypolymer substrate using an automatic spotting machine (DR. Easy spotter,Maio-Li, Taiwan), and immobilized by a UV crosslinker (STRATAGENE UVStratalinker 1800, Santa Clara, USA) with 0.24 J. The hybridizationreaction between each DNA template and probe was carried out with DR.Chip DIY Kit (DR. Chip Biotech, Maio-Li, Taiwan). The proceduresfollowed the manual and are briefly described below. The PCR product wasdenatured at 95° C. for 10 min, and cooled in an ice bath for 2 min. Tothe microarray chamber was added 200 ml of Hybridization Buffer(containing the 50 end-biotinylated oligonucleotide complementary to thesequence of positive control probe) and 1 μl of denatured PCR product,incubated at 47° C. with vibration for 1 hr, and washed three times withWash Buffer. The blocking reaction was then performed by mixing 0.2 mlof Strep-AP (Streptavidin conjugate alkaline phosphatase) and 200 ml ofBlocking Reagent at room temperature for 30 min, and washing three timeswith Wash Buffer. The colorimetric reaction was then implemented byadding 4 ml of NBT/BCIP and 196 ml of Detection Buffer in the chamber,developing in the dark at room temperature for 20 min, and washing twicewith distilled water. The hybridization result as shown in FIG. 1 wasindicated as the developed pattern on the microarray, which was readdirectly with the naked eyes.

As FIG. 1 shows, fifteen H5 AIVs, including ten LPAIVs and five HPAIVs(Table1) were tested using oligonucleotide microarrays following thePCR. All viruses were unambiguously detected and pathotyped, and nocross-reactions were found (FIGS. 1 B and C). The microarray resultswere completely concordant with the results of direct sequence analysisof the H5 gene spanning the CS region (Table 3).

The microarray detection results of artificial oligonucleotides areshown in FIG. 1 D. The nine representative AIV H5 oligonucleotides whichpossess five or more basic amino acids at the X displayed positive atthe universal probe dots, either at U1 or U2, or both. No cross-reactionwith other probes which detected one to four basic amino acids at X wasfound. This demonstrated good probe specificity of the probes. The totaldesigned 13 probes could successfully differentiate the number of basicamino acids and recognize the HPAIVs which possess five or more basicamino acids at the X of the CS PQ^(m)-X^(n)-*GLF (SEQ ID NO: 51) motif.The hybridization signals on microarrays indicated by colorimetry in thepresent invention made the results clearly identifiable using the nakedeyes, that is, no additional imaging equipment was needed here. Thisfinding shows that the simultaneous detection and pathotyping of AIV H5viruses can be inexpensively and easily achieved using oligonucleotidemicroarrays.

[Detection Limit]

The plasmid DNA with successful insert was extracted and quantified witha spectrophotometer (WPA UV1101, Biochrom Ltd, Cambridge, UK). The copynumbers of the DNA were calculated and the DNA was then diluted seriallyin TE buffer. The sensitivity of agarose gel and oligonucleotidemicroarray was investigated and compared by testing 10-fold serialdilations of DNA (10⁷ to 10⁰ copies) originating from the AIV referencestrains.

The sensitivity comparison test between the agarose gel assay andoligonucleotide microarray using A/CK/Hsinchu/A1939/11 (LV7) as anexample is shown in FIG. 2. The results showed that DNA below 4.6×10²copies/μL could not be seen on agarose gel. However, DNA with 4.6copies/μL could still be read on the microarrays. This indicated thatthe sensitivity of oligonucleotide microarray sensitivity was about 100times higher than that of the agarose gel assay.

The oligonucleotide microarray assay described in the present inventionoffers a simple, rapid and accurate approach to discriminate betweenLPAIVs and HPAIVs. This method provides new opportunities for avianinfluenza surveillance and diagnostics and may be particularlyattractive for large-scale screening of suspected AIV H5 viruses duringoutbreaks for regional diagnostic laboratories.

While example embodiments have been disclosed herein, it should beunderstood that other variations may be possible. Such variations arenot to be regarded as a departure from the spirit and scope of exampleembodiments of the present application, and all such modifications aswould be obvious to one skilled in the art are intended to be includedwithin the scope of the following claims.

What is claimed is:
 1. A probe selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO:
 13. 2. A kit for pathotyping of H5 avian influenza virus, comprising the probe defined in claim
 1. 3. The kit of claim 2, wherein the probe is further added a tail composed of 19 T bases and is spotted to a microarray substrate.
 4. The kit of claim 3, further comprising an oligonucleotide of SEQ ID NO: 14 spotted on the microarray substrate as a positive control probe.
 5. A method for pathotyping of H5 avian influenza virus in a sample, comprising: (a) obtaining a nucleic acid from the sample; (b) hybridizing the nucleic acid with a probe selected form the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; and (c) pathotyping the sample as a low-pathogenic avian influenza virus (LPAIV) or highly pathogenic avian influenza virus (HPAIV) based on the hybridization result.
 6. The method of claim 5, wherein the nucleic acid is obtained by extracting RNA from the sample, reverse transcribing the RNA into a DNA, and amplifying the nucleic acid in the DNA by a PCR reaction.
 7. The method of claim 6, wherein a set of primers of SEQ ID NO: 15 and SEQ ID NO: 16 is used in the PCR reaction.
 8. The method of claim 7, wherein the set of primers is 5′ end-biotinylated.
 9. The method of claim 5, wherein the nucleic acid further comprises a label.
 10. The method of claim 9, wherein the label is a fluorescent label, a chemiluminescent label, a colored dye label, a radioactive label, a radiopaque label, a protein including an enzyme, a peptide or a ligand.
 11. The method of claim 5, wherein the H5 avian influenza virus is a highly pathogenic avian influenza viruses (HPAIV) when the nucleic acid is hybridized to the probe selected from SEQ ID NO: 12 and SEQ ID NO: 13 or the nucleic acid is hybridized only to the probe of SEQ ID NO: 1 or SEQ ID NO: 2, or both when all the probes are used.
 12. The method of claim 5, wherein the H5 avian influenza virus is a low pathogenic avian influenza viruses (LPAIV) when the nucleic acid is hybridized to the probe selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO:
 11. 